Method of Producing Reduced Coenzyme Q10

ABSTRACT

The present invention relates to a method of producing high-quality reduced coenzyme Q 10  converted from oxidized coenzyme Q 10  by natural reductase. It is stable, completely natural and can be used on injection. This method is suitable for large-scale industrial production without special protective environment/atmosphere. The method of producing reduced coenzyme Q 10  includes three stages: {circumflex over (1)} phosphorylation of oxidized coenzyme Q 10  {circumflex over (2)} reduction of phosphorylated oxidized coenzyme Q Q 10  by biological reductase {circumflex over (3)} extracting reduced coenzyme Q 10  from reductases.

FIELD OF THE INVENTION

In the present invention, it is about producing method of reducedcoenzyme Q₁₀, especially about method of coenzyme converting to producereduced coenzyme Q₁₀.

BACKGROUND OF THE INVENTION

Coenzyme Q₁₀, also known as ubiquinone, is an oil-soluble orange crystalat room temperature, melting point 48° C., no smell, the structure issimilar to Vitamin K. Q refers to the quinone chemical group, and 10refers to the isoprenyl chemical subunits, the structure is asfollowing:

Coenzyme Q₁₀ has a another form of reduced Coenzyme Q₁₀, whosephysiological functions derive from the oxidizing and reducingcharacteristics of benzoquinonyl and physical characteristics ofisoprenoid side chain. It has two main functions on human being: first,strong effect of anti-lipid per oxidation; second, it plays an importantrole during the process of energy transformation from nurishments inmitochondrion. Quinone ring transfers electron and hytron in reductionbreathing chain. This function is essential for lives and vital for ATP.Reduced Coenzyme Q10 is a natural cell antioxidant and metabolismactivator. Meanwhile, it protects and resumes Integrality of biomembranestructure, stabilize membrane potential and improve body's non-specialimmunity. It is used as assistant treatment in heart and liver clinicalcuring.

It is Coenzyme Q₁₀which has the two effects as mentioned above.Therefore, producing Coenzyme Q₁₀ especially natural Coenzyme Q₁₀ willprovide a direct effect, However, Coenzyme Q₁₀ will easily get reducedand is difficult to achieve industrial production. Reduce Coenzyme Q₁₀ awhite crystal two-reduction from Coenzyme Q₁₀. It is known reduceCoenzyme Q₁₀ can be obtained by using reducing agent reduction onCoenzyme Q₁₀, such as WO01/52822A1. There is a problem of moleculereduction on case reduce Coenzyme Q₁₀, which is reduced by chemicalreducing agent, is used on healthy food, beverage, cosmetic and drugs.Up to now, commercial reduce Coenzyme Q₁₀ is not able to manufactured.Additionally, we have to solve others problems like how to protect andstabilize reduce Coenzyme Q₁₀.

Furthermore, those ,which rise environment polluting and are uncertainlysafe to human health, like chemical reducing agents, protestants andadditives, organic solvents , are widely used on producing reduceCoenzyme Q₁₀, exists in cosmetic ,food and drugs. The harm of reducingagent and protestant is even more than reduced Coenzyme Q_(10's) effect.

SUMMARY OF THE INVENTION

In view of the foregoing, the present invention is targeted to offer anenzymatic conversion method of producing reduced Coenzyme Q10 which isvery natural, green, safe and reliable.

The present invention contains following techniques: {circumflex over(1)} phosphorylation of oxidized coenzyme Q₁₀ {circumflex over (1)}reduction of phosphorylated oxidized coenzyme Q₁₀ by biologicalreductase {circumflex over (3)} extracting reduced coenzyme Q₁₀ fromreductases. The foregoing reaction formula is as:

The method according to stage 2, wherein it is preferred that:

-   -   (1) Choosing bacterial, microzyme, or epiphyte which contains        coenzyme Q₁₀ to develop.    -   (2) Disrupting cells, and deposit them with ammonium sulfate of        30%-70% weight percentage.    -   (3) Isolating the precipitate, dialysis the foregoing to get        biological reduced coenzyme Q₁₀.

The above biological reductase and phosphorylation oxidized are of finereduction in a higher yield.

As preferred, the microzyme or epiphyte is hotosynthetic bacteria or redPseudomonas.

As preferred, the cells are disrupted by Ultrasonic Cell Disruptor.

As preferred, washing precipitate in 35° C.˜45° C. cold water for 1˜2hours after deposition, and centrifugating it with Cooling centrifugesfor 10˜30 minutes under 1° C.˜3° C., 10000˜15000 r/min atmosphere, thencast supernatant, adding phosphoric acid solution to dialysis.

As more preferred, Sepharos 4B is added to stir after dialysis, andwashing three times with phosphoric acid solution, added with sodiumhydroxide to get a PH 8.5, breakdown, freeze drying the liquid afterfiltering with cool drying machine to obtain foregoing coenzyme Q₁₀.

As preferred, the phosphorylation of oxidized coenzyme Q₁₀ is carriedout by dissolving the oxidized coenzyme Q₁₀ in phosphoric acid solution,then adding phosphonolipide or any other phosphonolipide salt in anamount of 2-10% weight of oxidized coenzyme Q₁₀ with 10˜120minutesstirring under 28° C.˜45° C. atmosphere.

As more preferred, wherein the phosphoric acid solution PH range shouldbe adjusted to be 6.0˜8.2.

As preferred, the extraction from film pressed filtering reactants is ofvacuum to be concentrated from film pressed filtering with adding waterduring the concentration. Crystallization under 2° C.˜10° C. , thencooling and drying the crystalline to obtain the foregoing reducedcoenzyme Q₁₀.

As more preferred, the film pressure of filtering is 0.1 MP˜0.8 MP

As above motioned method of natural enzymatic conversion oxidizedcoenzyme Q₁₀, it is able to manufacture in a large-scale without specialprotective atmosphere. The foregoing obtained reduced coenzyme Q₁₀, ownsthe following features:

{circumflex over (1)} Stability. No reducing by dioxygen or any chemicalcompound. Coenzyme Q₁₀ remains its qualities in phosphoric acid solutionof PH6.0˜8.2, and has a shelf life of over 2 years below 50° C.

{circumflex over (2)} Full nature. It is completely safe to use on food,drug and cosmetic while no reducing agent, antioxidant or protectant areused during the process.

{circumflex over (3)} Water-solubility. It can be used on injectionwhich is completely water soluble after phosphorylation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is as HPLC prints of the present invention of reduced coenzymeQ₁₀.

DETAIL DESCRIPTION OF THE INVENTION EXAMPLE 1

Method of using bacteria to produce biological reductase:

1. Seed selection: Rhodobacter sphaeroides, a healthy sporty bacteriaseed of 0.5-0.9 micron width, 1.2-2.0 micron length, gammae, no capsule,single polar flagellum.

2. Preparing zymotic fluid: 5.5 g monometallic sodium orthophosphate',1.5 g Dlmalic acid , 2.0 g sodium acetate, 2.0 g sodium hydroxide, 1.0 gammonium chloride, 0.25 g magnesium chloride, 0.05 g calcium chloride,3.5 g glucose dissolved in 1.0 L distilled water, stirring PH range isbetween 6.5-7.0.

3. Prepare 10 triangular flasks of 250 ML, pouring 100 ML inoculum intoeach flask after sterilization. Seal them after inoculation. Put theminto incubator for 72 hours in sunlight of 32° C., and then get out.

4. Collecting inoculum, centrifugating it with centrifuge for 20minutes, 6000 r/min, then disrupt the mycelium after centrifugation,adding 200 ml brine of 3% weight percentage in still for 1 hour. thencollect supernatant, adding 500 ml ammonium sulfate, washed by 40° C.water for 1.5 hour, next centrifugation with cooling centrifuge for 20minutes, 12000 r/min at 4° C. Then cast supernatant, adding 300 MLphosphoric acid solution to dialysis, stirring with 100 ml Sepharos 4B,washed three times with 200 ml phosphoric acid solution. Adding sodiumhydroxide to adjust to PH8.5, break downing, cool drying the filteredliquid with cooling drying machine to obtain 80 mg biological reductase.

EXAMPLE 2

Method of using microzyme to produce biological reductase:

1. Seed selection: Schizosaccharomyces Promb.

2. Preparing zymotic fluid: beef extract 0.3 g, peptone 1.0 g, Nacl 0.5g, water 100 ML, adjust Phto 7.0˜7.2, adding water into breaker , weighup beef extract, peptone and Nacl, warm them up to be melted, adjust thePH to 7.0˜7.2, packing each with cotton tampon, then high pressuresteaming sterilizing to finish.

b 3. Preparing 10 triangular flasks of 250 ML, pouring 100 ML inoculuminto each flask after sterilization. Seal them after inoculation. Putthem into incubator for 72 hours in sunlight of 35° C., and then getout.

4. Collecting inoculum, centrifugating it with centrifuge for 20minutes, 6000 r/min, then disrupt the mycelium after centrifugation,adding 200 ml brine of 3% weight percentage in still for 1 hour, thencollect supernatant, adding 500 ml ammonium sulfate, washed by 40° C.water for 1.5 hour, next centrifugating with cooling centrifuge for 20minutes, 12000 r/min at 5° C. Then cast supernatant, adding 300 MLphosphoric acid solution to dialysis, stirring with 100 ml Sepharos 4B,washed three times with 200 ml phosphoric acid solution. Adding sodiumhydroxide to adjust to PH8.5, break downing, cool drying the filteredliquid with cooling drying machine to obtain 80 mg biological reductase.

EXAMPLE 3

Method of using fungus to produce biological reductase:

1. Seed selection: Entophytic

2. Preparing zymotic fluid: beef extract 0.3 g, peptone 1.0 g, Nacl 0.5g, agar 1.5 g, water 100 ML.

3. Preparing 10 triangular flasks of 250 ML, pouring 100 ML inoculuminto each flask after sterilization. Seal them after inoculation Putthem into incubator for 72 hours in sunlight of 36° C., and then getout.

4. Collecting inoculum, centrifugating it with centrifuge for 20minutes, 6000r/min, then disrupt the mycelium after centrifugation,adding 200 ml brine of 3% weight percentage in still for 1 hour. thencollect supernatant, adding 500 ml ammonium sulfate, washed by 40° C.water for 1.5 hour, next centrifugating with cooling centrifuge for 20minutes , 12000 r/min at 2° C. Then cast supernatant, adding 300 MLphosphoric acid solution to dialysis, stirring with 100 ml Sepharos 4B,washed three times with 200 ml phosphoric acid solution. Adding sodiumhydroxide to adjust to PH8.5, break downing, cool drying the filteredliquid with cooling drying machine to obtain 80 mg biological reductase.

EXAMPLE 4

Using oxidized coenzyme Q₁₀ to react with reductase, converting intonatural reduced coenzyme Q₁₀.

1. phosphorylating the oxidized coenzyme Q₁₀, then dissolve 250 goxidized coenzyme Q₁₀ in 1000 ML phosphoric acid solution((PH7.4), thenadding 12.5 g phosphonolipide in an amount of 5% weight of oxidizedcoenzyme Q₁₀ with 10˜120 minutes stirring under 37° C. atmosphere.

2. Prepare biological reductase according to example 1˜3.

3. Put the phosphorrlated oxidized into reactor, adding 80g biologicalreductase, stirring for 10˜30minutes at 37° C.˜60° C. atmosphere

4. Film filtering: with a pressure of 0.5 mp, making filtering at 30°C., and leave the filtrate in the air for above 5 hours at 2° C.

5. Concentrating the filtrate at 35° C. , 200 Pa vacuum atmosphere.

6. Washing the concentration for 3 times with water, and thencrystallizing at 2° C.

7. Obtaining 248.2 g crystal in the cooling drying machine, the purityreaches 99.4%. As tested, no oxidized coenzyme Q₁₀ exists.

Testing Example

Prepare reduced coenzyme Q₁₀ according to example 4, test it with HPLC.Equipments: Shimazu wave length: 275 nmMobile phase: enthanol:methanol=1:1 (v:V)Column: octadecyl silica gel column; length :180 mm inner diameter: 3.2mmWeigh up reduced coenzyme Q₁₀20 mg, dissolved in enthaol to 100 ml, sample size 20 ul, usingthree-point external standard method to calculate assay.Appearance time 8.974 min. Tested chart as Chart 1.

1. A method of producing reduced coenzyme Q₁₀ including: followingsteps: {circumflex over (1)} phosphorylation of oxidized coenzyme Q₁₀{circumflex over (2)} reduction phosphorylated oxidized coenzyme Q₁₀ bybiological reductases {circumflex over (3)} extracting reduced coenzymefrom reductases.
 2. The method according to claim 1, wherein thebiological reductases are made by: (1) choosing bacteria, microzyme orepiphyte containing coenzyme Q₁₀ and cultivating the bacteria, microzymeor epiphyte containing coenzyme Q₁₀. (2) disrupting cells cultivatedfrom step (1), and deposit them with ammonium sulfate in a concentrationof 30%-70% weight percentage to get a precipitate. (3) isolating theprecipitate gotten from (2) to get biological reduced coenzyme Q₁₀ byusing dialysis.
 3. The method according to claim 2, wherein thebacteria, microzyme or epiphyte is hotosynthetic bacteria or redpseudomonas.
 4. The method according to claim 2, wherein the cells aredisrupted by aultrasonic cell disruptor.
 5. The method according toclaim 2, 3 or 4 wherein wash the precipitate in 35° C.˜45° C. cold waterfor 1˜2 hours after the depositing , then centrifugate precipitate withcooling centrifuges for 10˜30 minutes under 1° C.˜3° C., 10000˜15000r/min atmosphere, then cast supernatant, add phosphoric acid solution todialysis.
 6. The method according to claim 5, wherein sepharos 4B isadded to stir after dialysis, then wash three times with phosphoric acidsolution, add sodium hydroxide to get a PH 8.5, breakdown, freeze drythe liquid after filtering with cool drying machine to obtain coenzymeQ₁₀.
 7. The method according to claim 1 or 2, wherein thephosphorylation of oxidized coenzyme Q₁₀ comprises steps: dissolve theoxidized coenzyme Q₁₀ in phosphoric acid solution, then addingphosphonolipide or phosphonolipide salt in an amount of 2˜10% weight ofthe oxidized coenzyme Q₁₀ with 10˜120minutes stirring under 28° C.˜45°C. atmosphere.
 8. The method according to claim 7, wherein thephosphoric acid solution is in PH 6.0˜8.2.
 9. The method according toclaim 1 or 2, wherein the extraction from film pressed filteringreactants is vacuumly concentrated with adding water during theconcentration, then make crystallization under 2° C.˜10° C. , thencooling and drying the crystalline to obtain the reduced coenzyme Q₁₀.10. The method according to claim 9, wherein the film pressure offiltering is 0.1 MP˜0.8 MP